The goal of this research proposal is to develop a potent and safe vaccine for protection against Venezuelan Equine Encephalitis (VEE). Several attenuated VEE strains have been generated for the purpose of creating a prophylactic vaccine, including a strain called TC-83 which has been licensed for use in horses. However, these attenuated strains have either proven to be too dangerous to use in humans or have a difficult regulatory approval process in front of them before they will reach the market due to the neuraltropism of the VEE virus. In contrast, using a safer and well-characterized viral vector to deliver the key antigens of VEE does not have these same regulatory issues. Published experiments have demonstrated that a recombinant adenovirus expressing the E2, E3, and 6K genes (E2E36K) of TC-83 can afford protection against aerosolized VEE in animal models. The problem is simply efficiency;the amount of recombinant adenovirus necessary for protection is prohibitively large to be effect in humans. Using West Coast Biological's patent pending technology, preliminary studies in this proposal demonstrate an expressed Toll Receptor 3 ligand, given in conjunction with antigen expressed from a non-replicating adenovirus vector significantly augments the adaptive immune response, and can improve antibody responses to the transgene by two orders of magnitude. The proposed Aims tests whether the combination of the VEE antigen with WCB's expressed TLR3 vector can sufficiently and practically improve immunogenicity and protection against VEE. Aim 1 of this proposal strives to determine whether expressing TLR3 ligand and E2E36K antigen in an adenoviral vector can improve antibody responses to VEE over standard rAd expressing the E2E36K antigen. Aim 2 determines whether the route of delivery of the expressed TLR3 ligand vector can improve systemic and mucosal immune responses, and whether pre-existing immunity to adenovirus can be circumvented by use of mucosal routes of delivery. Aim 3 investigates the ability of the expressed TLR3 ligand vector with E2E36K antigen to protect against virulent Trinidad Donkey strain VEE and other more heterologous strains. For all of these aims, we will measure antibody responses in mice using ELISA based assays. Together, these aims will map the development plan to commercialize the vaccine. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: West Coast Biologicals (WCB) is developing a new vaccine to prevent infection of Venezuelan Equine Encephalitis (VEE), a pathogen that has a high incidence of accidental laboratory exposure and that has been explored as a biowarfare agent in the past. Because of the dangers involved with some of the previous vaccine strains, WCB has made a safer vector, based upon our patent pending technology, and will explore the ability of our research vector to protect against VEE in well-characterized animal models.